Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.     

Orientation and molecular map position of the complement genes in the mouse MHC.

Over the past few years six gene clusters have been isolated from the major histocompatibility complex (MHC) of the BALB/c mouse encompassing a total of 1600 kb of DNA and 48 genes. The molecular distances between these gene clusters and the orientation of four of the six clusters on chromosome 17 is not known. Here we use pulse-field gradient gels and Southern blot hybridization to establish large-scale genomic restriction maps covering several hundreds of kb surrounding the three gene clusters located in the K, I, S, and D regions of the MHC. Comparison of the maps orients the complement gene clusters in the S region with the 21-OHB gene pointing towards the K end and the C2 gene pointing towards the D end of the MHC. The distances between the E alpha and 21-OHB genes is 430 kb and between the C2 and TNF-alpha genes at least 420 kb.     

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass

The effect of oxygen on N₂-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 μM O₂, some of them being carbon limited. All strains needed a minimum amount of oxygen for N₂-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O₂ concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N₂ fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N₂ fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O₂ concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Preface

Goldman Festschrift

Preface     

Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer

The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Odorant response of isolated olfactory receptor cells is blocked by amiloride

Summary Olfactory receptor cells were isolated from the nasal mucosa ofRana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1)On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 m amiloride added to the odorant solution. With 10 m amiloride, the response to odorants was only partially abolished. 2)Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between –80 and –90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida,Polychaeta)

Summary Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to: A.W.C. Dorresteijn